Inordinate Spinescence: Taxonomic Revision and Microtomography of the Pheidole cervicornis Species Group (Hymenoptera, Formicidae)

Figures

Abstract

The ant genus Pheidole—for all of its hyperdiversity and global ubiquity—is remarkably conservative with regard to morphological disparity. A striking exception to this constrained morphology is the spinescent morphotype, which has evolved multiple times across distantly related lineages of Indoaustralian Pheidole. The Pheidole cervicornis group contains perhaps the most extraordinary spinescent forms of all Pheidole. Here we present a taxonomic revision of the P. cervicornis group, and use microtomographic scanning technology to investigate the internal anatomy of the thoracic spines. Our findings suggest the pronotal spines of Pheidole majors, are possibly skeletomuscular adaptations for supporting their disproportionately large heads. The ‘head support hypothesis’ is an alternative to the mechanical defense hypothesis most often used to explain spinescence in ants. The P. cervicornis group is known only from New Guinea and is represented by the following four species, including two described here as new: P. barumtaun Donisthorpe, P. drogon sp. nov., P. cervicornis Emery, and P. viserion sp. nov. The group is most readily identified by the minor worker caste, which has extremely long pronotal spines and strongly bifurcating propodeal spines. The major and minor workers of all species are illustrated with specimen photographs, with the exception of the major worker of P. cervicornis, which is not known.

Data Availability: All relevant data are within the paper and its Supporting Information files. The full volumetric datasets have been archived at the Dryad Data Repository (http://datadryad.org, doi: 10.5061/dryad.1j41m).

Funding: This work was supported by subsidy funding to OIST and NSF (DEB-1145989).

Competing interests: The authors have declared that no competing interests exist.

Introduction

The global rise of the hyperdiverse ant genus Pheidole is an evolutionary epic with many subplots [1, 2]. One of the most intriguing of these is the evolution of highly adorned “spinescent” Pheidole. This extreme morphotype is represented by a small minority of spiny outliers within a genus not otherwise known for morphological disparity [3, 4]. Although the genus originated in the Americas, the massive radiation of New World Pheidole do not include any extant spinescent species. Rather, spinescent Pheidole are enigmatically confined to the Old World Indo-Australian tropics. The only exceptions to this rule are the fossil taxa P. tethepa Wilson and P. primigenia Baroni Urbani described from Dominican amber [5, 6]. Once classified under the single subgenus Pheidolacanthinus, recent phylogenetic studies revealed that spinescent Pheidole evolved independently in four independent Indoaustralian lineages [1, 7]. The evolution of spinescent Pheidole and associated ecological changes has been studied in the context of evolutionary biogeography and the taxon cycle hypothesis [7–9]. The ecological significance of spinescence, however, remains unexplained.

Here we present our research on the most spinescent of all 1000+ Pheidole known to science. We used 3D X-ray microtomography to illustrate these extreme phenotypes, and to investigate the functional morphology of spinescence. Micro-ct is an emerging tool for taxonomic (e.g. [10, 11]) and functional anatomy studies (e.g. [12]). We used microtomography to test the hypothesis that pronotal spines in Pheidole majors are a skeletomuscular adaptation for supporting disproportionately large heads. While the oversized heads and massive mandibles of Pheidole majors can serve as effective colony defense, they are used by many species as biological millstones for processing seeds too hard for minor workers to crush [2, 13–17]. A comparative study of thorax architecture among ants recently revealed that, in contrast to queens, workers possess enlarged neck muscles in the first thoracic (T1) segment [18]. These expanded thoracic muscles are the modification that allows worker ants to lift and carry objects many times heavier than themselves. Presumably, the T1 muscles also allow Pheidole majors to support their massive head capsules and dense mandibles. If the thoracic muscles extend into the pronotal spine cavity, the extra capacity and transverse angle could increase head support. Alternatively, if the pronotal spines are devoid of muscle fibers, their function is unrelated to head support.

The subject of this study, the Pheidole cervicornis group (Formicidae: Myrmicinae), consists of four remarkably spinescent species (two of which are described here for the first time) that are endemic to mid-and high-elevation forests of New Guinea. The group was erected by Emery [19] to house the single species P. cervicornis Emery. Wilson [20] and Sarnat [21] both proposed that the cervicornis group was closely related to the spinescent Electropheidole (= Pheidole roosevelti group) of Fiji. However, subsequent phylogenetic analyses demonstrated that these two groups are only distantly related to each other, and that spinescence has evolved independently in each species clade [1, 7]. The cervicornis group is putatively monophyletic [1] and sister to a much larger radiation of less spinescent species in the P. bifurca group. These two sister groups are descended from a major clade of Pheidole that have radiated throughout New Guinea and Australia.

The following study is a small contribution towards understanding the evolutionary landscape that precipitated a handful of distantly related Pheidole lineages to converge towards a shared phenotype of extreme spinescence. Unlike the celebrated Anolis lizards of the Caribbean [22] and the Cichlid fishes of African lakes [23]—or even the spiders of Hawaii [24]—a substantial amount of work remains to be done on the basic taxonomy, morphology and ecology of Indoaustralian Pheidole before sufficient data is available for testing theories of convergent evolution, adaptive radiation, and morphological innovation. In the future, we hope to extend the revisionary and anatomical work presented here for the P. cervicornis group to the other major radiations of spinescent Pheidole.

Methods

X-ray microtomography

X-ray microtomography scans were taken with a Zeiss Xradia 510 Versa scanner and the included XMController software. XMReconstructer software was used post-imaging for 3D reconstruction and output files were saved in dicom format. Scanning preparation of dry mounted ant specimens on paper points only involved gluing the point including the ant to a short piece (~4cm) of MicroLumen high performance medical tubing. Parameters for all scans were chosen according to scanning directions in the Xradia 510 Versa manual. Images were taken with a 4x objective, binning of two by two pixels. Other scan parameters typically varied from 30 to 65 keV, 2 to 5.5 W. Exposure times were between 1 and 15 seconds (Table 1). Full 360 degree rotations were done with 1601 projections. The resulting scans have a resolution of typically 1013x992x993 (HxWxD) pixels and voxel sizes range from 0.00286 to 0.00549 mm.

Rotation videos were created using Amira software from Volren data visualizations by creating a Camera Path object with seven key frames (rotating around the three standard views: profile, full-face, and dorsal view), selecting constant rotation speed, and saving the movie files in mpeg format with 480p resolution, 24 frames per second frame rate and selecting AntiAliasing2 in the movie maker function. The number of mesh triangles was reduced with a three-step approach for creating the 3D PDFs. First, the freely available software Meshlab (where the scan data were imported as STL files) was used to select and manually remove the paper points bearing the specimens. The scans were cleaned from small artifacts and other isolated fragments (Filters > Remove isolated piece (wrt diameter) (diameter size = 1%). Second, the specimens’ interior structure was removed by selecting the functions: (1) Filters > Color Creation and Processing > Ambient Occlusion Per Vertex, (2) Filters > Selection > Select Faces By Vertex Quality (min = 0, max = 0.001), (3) Remove Selected Faces. This sequence isolates the visible vertexes representing the specimens’ exterior. Third, the amount of vertexes was reduced to slightly less than 1 million mesh triangles (Filters > Quadratic Edge Collapse Decimation). The resulting STL files, each between 200–300 megabytes, were opened in Adobe Acrobat Pro, using the Tetra4D converter app, where they were annotated and saved as 3D PDFs.

Morphometric measurements and illustration

External morphological characters were quantified and are reported as lengths or indices. Measurements were made with a stereomicroscope at 40x magnification using a dual-axis stage micrometer wired to digital readouts. Digital color specimen photographs were taken using the Auto-Montage software package (Syncroscopy) in combination with a JVC KY-F7U digital camera mounted on a Leica MZ16 dissecting scope, and the software package Helicon Focus in combination with a Leica DFC450 digital camera mounted on a Leica M205C dissecting scope. Vector illustrations were made in Adobe Illustrator by tracing specimen photographs.

Morphometric measurements were recorded in thousandths of millimeters, but are reported here to the nearest hundredth as a range from minimum to maximum across all measured specimens. Specimens for measurements were chosen to reflect potential morphological variation across the full geographic range. The number of specimens from which measurements were taken for a given caste is referred to by n. Various measurements and morphological terms used here are illustrated in Sarnat (21, Figs 1–7). All specimen data and specimen images are available on the Antweb.org website, and all nomenclatural and taxonomic changes are recorded on Antcat.org website.

Measurement and indices abbreviations

EL Eye length. Maximum measureable eye length.

FL Metafemur length. Length of metafemur measured along its long axis.

HL Head length. Maximum distance from the midpoint of the anterior clypeal margin to the midpoint of the posterior margin of the head, measured in full-face view. In majors, measured from midpoint of tangent between anteriormost position of clypeus to midpoint of tangent between posteriormost projection of posterolateral lobes.

HW Head width. Maximum width of the head in full-face view, excluding the eyes.

ML Mesosomal length. Measured in lateral view as the diagonal length of the mesosoma from the point at which the pronotum meets the cervical shield to the apex of the propodeal lobe.

PSL1 Pronotal spine length. Length of pronotal spine measured in dorsal-oblique view such that the base and apex of the spine are in the same focal plane. For P. cervicornis measured from middle of trunk to most apical point of posterior prong.

PSL2 Propodeal spine length, vertical portion. For major workers the length of the entire propodeal spine measured in profile view from center of propodeal spiracle to apex. For minor workers measured in profile view from the center of propodeal spiracle to the dorsal junction of the anterior and posterior projections.

PSL3 Propodeal spine length, horizontal portion. For minor workers the straight-line distance between the apices of the anterior and posterior projections of the propodeal spine, measured in profile.

SL Scape length. Length of the antennal scape, including the lamella encircling the base of the scape but excluding the basal condyle.

CI Cephalic index. HW/HL × 100.

SI Scape index. SL/HW × 100.

Museum abbreviations

BMNH The Natural History Museum (London, United Kingdom)

HNHM Hungarian Natural History Museum (Budapest, Hungary)

USNM United States National Museum of Natural History (Washington D.C., USA)

Nomenclatural acts

The electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature, and hence the new names contained herein are available under that Code from the electronic edition of this article. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix “http://zoobank.org/”. The LSID for this publication is: urn:lsid:zoobank.org:pub: 10D51A80-CD4F-4EDA-A755-FC9ADD3C81EF. The electronic edition of this work was published in a journal with an ISSN, and has been archived and is available from the following digital repositories: PubMed Central, LOCKSS [author to insert any additional repositories].

Results and Discussion

X-ray Microtomography

Taxonomic illustration, interactive 3D PDF’s and rotation videos.

Volumetric surface models of the minor workers of each species (Fig 1) are presented to illustrate the extreme morphologies that have evolved within the Pheidole cervicornis group. Volumetric surface models of the major worker hypostomal bridge are presented to illustrate an important taxonomically and phylogenetically informative character used for diagnosing Pheidole clades (Fig 2). The three-dimensionality of this character is difficult to demonstrate using conventional methods, but is well-illustrated with micro-ct scans. Interactive 3D PDF’s of volumetric surface models were created for all available worker subcastes of the Pheidole cervicornis group species (Table 2).

Internal anatomy of thoracic spines.

X-ray micro-ct scans of a Pheidole drogon major worker specimen (Fig 3) and P. viserion (S8 Vid) reveal that the many branching muscle fibers of the first thoracic segment extend throughout substantial portions of the pronotal (anterior) spines. The pronotal spines' interior cuticle appear to serve as anchor points for muscles branches that converge anteriorly and connect to the head capsule. In contrast, the pronotal spines of the minor worker are hollow, aside from a few basal fibers (Fig 4). Moreover, the propodeal (posterior) spines of both major and minor worker of P. drogon lack substantial muscle fibers. The same musculature pattern was also observed for the other three species of the P. cervicornis group.

These findings, though preliminary, are consistent with the ‘head support hypothesis’ that pronotal spines of Pheidole majors are skeletomuscular adaptations for supporting their disproportionately large heads. Although a more comprehensive comparative study is necessary to rigorously test this hypothesis, these results offer the first alternative to the assumption that the spinescent architecture observed in ants functions primarily as mechanical defense

Contrary to the head support hypothesis of pronotal spines in Pheidole majors are the many Pheidole species whose majors exhibit comparably large heads but lack spinescent phenotypes. Moreover, pronotal spines are not observed in other ant lineages with specialized large-headed major workers. For example, both majors and minors of Carebara lack pronotal spines. Paradoxical to the head support hypothesis, pronotal spines occur in Acanthomyrmex minor workers but are lacking in the majors. Comparative studies, such as the one conducted by Paul and Gronenberg examining mandible muscles of ants [25], are required to further test the head support hypothesis. Future studies would benefit from the sampling of ant taxa both across spinescent and non-spinescent Pheidole lineages, and across other ant lineages with dimorphic worker castes.

Distribution and diagnosis of the Pheidole cervicornis species group

All known occurrences of species belonging to the Pheidole cervicornis-group are recorded from New Guinea (Fig 5). In addition to the morphological characters common to all Pheidole, the following characters diagnose the worker caste of the P. cervicornis group from those of all other congeners.

All four species within the Pheidole cervicornis group are immediately distinguished from the vast majority of congeners by the presence of extremely long pronotal spines and propodeal spines. The only other Pheidole clades with pronotal spines all occur in the Old World. These include the quadricuspis clade, the quadrispinosa clade and the bifurca clade. The quadricuspis group is restricted to Southeast Asia and does not occur as far east as New Guinea. The major workers are superficially quite similar to those of the cervicornis group, but are distinguished by the (1) lack of a central median hypostomal tooth, (2) coarsely rugoreticulate head sculpture, and (3) shorter, thicker and more abundant pilosity. The minor workers of the quadricuspis group are easily distinguished by the (1) propodeal spines, which are simple and very short (subequal to eye length), and (2) lack of mesonotal spines or lobes.

The quadrispinosa group is sympatric with the cervicornis group in New Guinea. In general, quadrispinosa group workers are smaller than those of the cervicornis group, with proportionally shorter limbs. The major workers as distinguished by the (1) lack of a central median hypostomal tooth, (2) coarsely rugoreticulate head sculpture, (3) deeply excavated antennal scrobes, (4) distinctly concave head vertex, and (5) relatively shorter and more triangular pronotal spines. The minor workers are easily distinguished by their smaller size and non-bifurcating propodeal spines (although they can be strongly curved).

The bifurca group is reciprocally monophyletic with the cervicornis group, and its constituent species are unsurprisingly most difficult to distinguish from those of the cervicornis group. The bifurca group is sympatric with the cervicornis group (and the quadrispinosa group) in New Guinea. Moreover, the bifurca group is represented by a dizzying number of undescribed species and morphological variation. Based on our preliminary survey of the bifurca group, the major workers are best distinguished from those of the cervicornis group by the (1) shorter pronotal spines, (2) less spinose or lobate mesonotal processes, and (3) smaller size. Additionally, the hypostomal bridge of most (but not all) bifurca group majors have a stout median tooth and either a pair of very weak or absent submedian teeth. However, there is at least one species in which the hypostomal arrangement is similar to that of the cervicornis group species (stout median tooth flanked by a pair of stout submedian teeth). The minor workers of the bifurca group are extremely variable with respect to length and shape of their mesosomal armaments. Some have bifurcating propodeal spines, some have very long pronotal spines, and some have mesonotal processes. None that we have examined, however, possess the combination of all three. Pheidole purpurascens Emery perhaps comes closest to the cervicornis group morphotype, but this species only has angled (versus bifurcate) propodeal spines.

The only Pheidole species outside of the aforementioned groups that has pronotal spines are P. aristotelis Forel and P. hainanensis Chen et al [26, 27]. However, the pronotal and propodeal spines of both species are only subequal in length to the eye, and the latter are not bifurcated. Moreover, the major worker lacks distinct pronotal spines.

The Pheidole roosevelti group [21] also deserves mention here. While its constituent members lack pronotal spines, many of the species have strongly bifurcating propodeal spines that strongly resemble those seen in the cervicornis group, in addition to mesonotal processes. These morphological similarities are entirely convergent, however, as the roosevelti group is only distantly related to the cervicornis group [1, 7].

The major workers of Pheidole barumtaun are separated from those of P. drogon by the fine carinulae that cover the posterolateral lobes. They are separated from P. viserion by the less sculptured gastral tergite, less striate mandibles and more arcuate carinulae of the frons. Both major and minor workers of P. barumtaun are separated from P. viserion by head and gaster color, which are dark brown in the former and clear yellow in the latter. The minor workers are separated from those of P. drogon by the narrower head (CI 84–90 vs. 89–91), longer legs (FL 1.22–1.38 mm vs. 1.07–1.16 mm) and relatively longer antennal scapes (SI 146–165 vs. 135–141).

There are, however, some distinct differences between the type series (Cyclops Mts.) and the material collected by Snelling from the Wapoga River Area. The type series workers, both majors and minors, are distinctly tricolored with dark brown heads and gasters that contrast with a yellowish mesosoma. The Wapoga workers are uniformly dark reddish brown. The type series majors also have downcurved propodeal spines, versus upturned in the Wapago material. However, there are not clear morphometric differences between these populations, and until further evidence is gathered we consider the variation to be infraspecific.

Pheidole barumtaun was named by Donisthorpe after Barumtaun camp where Cheesman made her collection. Among the examined material, workers were collected from the following microhabitats of primary montane forest: under loose bark of log, in and under living bark of recently felled tree, crown of Pandanus, decayed stalk in Pandanus, in leaf litter. Several of these microhabitats suggest the species might nest and forage in vegetation.

Pheidole cervicornis, known only from the minor worker subcaste, is the most distinct member of its eponymous group. The minor workers are the only members of the group which have bifurcated pronotal spines. They are also immediately recognizable by the thickly crenulated rugulae on the head and mesosoma. Additionally, the minor worker has a more circular head with a broad, weakly emarginated posterior margin (vs. elongate head with a narrow and flat posterior margin), and all the limbs are relatively shorter. There is some variation in color between specimens collected from western New Guinea (darker and have a contrasting white spot on their gaster), and eastern New Guinea (lighter and lacking white spot). We consider this infraspecific variation until additional collections prove otherwise. Aside from one nest collected from beneath a stone by Wilson (20), Pheidole cervicornis has only been recorded from stray foragers collected from the ground, in leaf litter, on logs, and in logs. It is the most widely collected species of the group, occurring along the northern coast of New Guinea, and also occupies the lowest elevation range (30–800 m). The species also appears to be more tolerant of disturbance than its close relatives, and has been collected in secondary and disturbed forest habitats.

The major workers of Pheidole drogon are distinguished from those of all other cervicornis-group species by the posterolateral lobe and gastral tergite, both of which are glossy and free of sculpture or shagreening. The minor workers are difficult to distinguish from those of P. barumtaun, and readers are referred to the discussion of that species for differentiating characteristics. The species is only known from the type locality where it was collected in montane rainforest on low vegetation and also recruiting to a tuna bait.

Etymology: The species name refers to Drogon, the black-colored dragon of Daenerys Targaryen, a fictional character from the George R. R. Martin’s novel A Song of Ice and Fire. The name is a noun in apposition and thus invariable.

Pheidole viserion is the only member of the cervicornis group that is uniformly yellow in color. The species is most similar to P. barumtaun, but the majors of P. viserion can be distinguished by the more striate mandibles and first gastral tergite, and cephalic sculpture. The minor workers are quite similar to those of P. barumtaun, and are best differentiated by the uniform yellow color.

Pheidole viserion has been collected from three sites in Papua New Guinea from montane primary forests and from a lowland habitat transitioning between primary and secondary forest. In addition to being found in the leaf litter, it was also found foraging in a hollow trunk above the ground.

Etymology: The species name refers to Viserion, the cream and gold colored dragon of Daenerys Targaryen, a fictional character from the George R. R. Martin’s novel A Song of Ice and Fire. The name is a noun in apposition and thus invariable.

Supporting Information

If viewed with Adobe Acrobat Reader (version 8 or higher), the interactive 3D-mode can be activated after trusting the document by clicking on the image, allowing the user to rotate, move and magnify the model.

If viewed with Adobe Acrobat Reader (version 8 or higher), the interactive 3D-mode can be activated after trusting the document by clicking on the image, allowing the user to rotate, move and magnify the model.

If viewed with Adobe Acrobat Reader (version 8 or higher), the interactive 3D-mode can be activated after trusting the document by clicking on the image, allowing the user to rotate, move and magnify the model.

If viewed with Adobe Acrobat Reader (version 8 or higher), the interactive 3D-mode can be activated after trusting the document by clicking on the image, allowing the user to rotate, move and magnify the model.

If viewed with Adobe Acrobat Reader (version 8 or higher), the interactive 3D-mode can be activated after trusting the document by clicking on the image, allowing the user to rotate, move and magnify the model.

If viewed with Adobe Acrobat Reader (version 8 or higher), the interactive 3D-mode can be activated after trusting the document by clicking on the image, allowing the user to rotate, move and magnify the model.

If viewed with Adobe Acrobat Reader (version 8 or higher), the interactive 3D-mode can be activated after trusting the document by clicking on the image, allowing the user to rotate, move and magnify the model.

Acknowledgments

Brian Brown for assisting with the loan of LACM material; Milan Janda and Phil Ward for loaning specimens; Michele Esposito and Brian Fisher for their support with Antweb. We thank John Deyrup, Cong Liu, Julia Janicki for assistance with CT-scanning and post-processing, Masako Ogasawara for help with specimens and imaging, and Kenneth Dudley for his assistance with GIS mapping. We thank Francisco Hita-Garcia and Xavier Espadaler for reviewing and improving an earlier manuscript of this publication.

Author Contributions

Conceived and designed the experiments: EMS GF EPE. Performed the experiments: EMS GF. Analyzed the data: EMS GF EPE. Wrote the paper: EMS GF EPE.

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